Method and compositions for producing berry derived products

ABSTRACT

A method for isolating a mixture of anthocyanins, bioflavonoids and phenolics from an edible berry using adsorbent resins which are regenerable for reuse is described. The mixture with a consumable carrier is particularly useful in foods and as a dietary supplement or nutraceutical product.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 09/383,324, filed Aug. 26, 1999, which is a Continuation-in-part ofU.S. application Ser. No. 09/317,310, filed May 24, 1999 now U.S. Pat.No. 6,423,365 Jul. 23, 2002, and this application also claim s priorityto U.S. Provisional Application Serial No. 60/120,178, filed Feb. 16,1999.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None

BACKGROUND OF THE INVENTION

(1) Summary of the Invention

The present invention relates to a method of preparation of edible berryderived compositions, particularly cherry, and to a method of use of thecompositions derived from the berries as phytoceutical/nutraceutcaldietary supplements or as an additive to foods. In particular, thepresent invention provides a natural berry composition, particularlycherry, containing a mixture of anthocyanins, bioflavonoids andphenolics for use as dietary supplements or as a food additive.

(2) Description of Related Art

Many plant-derived compounds may also impart important positivepharmacological or “nutraceutical” traits to foods by way of theirabilities to serve as cellular antioxidants by maintaining low levels ofreactive oxygen intermediates, as anti-inflammatory agents by inhibitingprostaglandin synthesis, or as inhibitors of enzymes involved in cellproliferation. These activities may be important in ameliorating chronicdiseases including cancer, arthritis, and cardiovascular disease(Kinsella et al., Food Tech. 85-89 (1993). Thus, with natural products,the dietary supplement/food industry and nutraceutical companies has theopportunity to employ compounds which can not only enhance foodstability as effectively as synthetic antioxidants, but can also offersignificant health benefits to the consumer.

Colorants like anthocyanins have been regarded as the index of qualityin tart cherries. Most importantly, recent results showed thatanthocyanins such as cyanidin-3-glucoside have strong antioxidantactivities (Tsuda, T., et al, J. Agric. Food Chem. 42:2407-2410 (1994)).The addition of antioxidants is one of the popular methods to increasethe shelf life of food products which is thought to be associated withlipid peroxidation. Natural antioxidants may play an important role inthe prevention of carcinogenesis. Dietary antioxidants may be effectiveagainst the peroxidative damage in living systems (Halliwell, B. and J.M. C. Gutteridge, Free radicals in biology and medicine. OxfordUniversity Press, New York 416-494 (1989); Osawa, T., et al, Role ofdietary antioxidants in protection against oxidative damage. Inantimutagenesis and anticarcinogenesis Mechanisms; Kuroda, Y.; Shankel,D. M., Waters, M. D., Eds.; Plenum Publishing. New York 139-153 (1990)).

Early studies have showed that MONTMORENCY cherry contains theanthocyanins cyanidin-3-gentiobioside and cyanidin-3-rutinoside (Li, K.C., et al., J. Am. Chem. Soc. 78:979-980 (1956)).Cyanidin-3-glucosylrutinoside was also found in six out of the sevensour cherry varieties (Harborne, J. B., et al., Phytochemistry 3:453-463(1964)). Dekazos (Dekazos, E. D., J. Food Sci. 35:237-241 (1970))reported anthocyanin pigments in MONTMORENCY cherry aspeonidin-3-rutinoside, peonidin and cyanidin along withcyanidin-3-sophoroside, cyanidin-3-rutinoside and cyanidin-3-glucoside.However, cyanidin-3-glucosylrutinoside as well as cyanidin-3-glucoside,cyanidin-3-sophoroside and cyanidin-3-rutinoside were identified as mainpigments in sour cherries. Using HPLC retention values, Chandra et al(Chandra, A., et al., J. Agric. Food Chem. 40:967-969 (1992)) reportedthat cyanidin-3-sophoroside and cyanidin-3-glucoside were the major andminor anthocyanins, respectively, in Michigan grown MONTMORENCY cherry.Similarly, cyanidin-3-xylosylrutinoside was detected as a minor pigmentin MONTMORENCY cherry (Shrikhande, A. J. and F. J. Francis, J. Food Sci.38:649-651 (1973)).

In the prior art, production of pure anthocyanins (compounds 1-3 ofFIG. 1) from BALATON and MONTMORENCY cherry juices was carried out firstby adsorbing the pigment on an AMBERLITE XAD-2 (Sigma Chemicals) column(Chandra, A., et al., J. Agric. Food Chem. 41:1062-1065 (1993)). Thecolumn was washed with water until the eluant gave a pH of approximately7.0. The adsorbed pigments along with other phenolics were eluted withMeOH. The resulting crude anthocyanins were fractionated and purified byC-18 MPLC and HPLC, respectively, to afford pure anthocyanins forspectral studies. Purification of 500 mg crude MONTMORENCY anthocyaninsfrom AMBERLITE XAD-2 yielded 60 mg of pure anthocyanins 1-3 compared to391.43 mg from BALATON. This research indicated that crude anthocyaninsfrom MONTMORENCY obtained from the XAD-2 contained a high percentage ofother organic compounds. The AMBERLITE XAD-2 did not allow recycling ofthe resin. There was no attempt to use the crude mixture of phenolicsand anthocyanins for any purpose. U.S. Pat. No., 5,266,685 to Garbutt,U.S. Pat. No. 5,665,783 to Katzakian et al and U.S. Pat. No. 5,817,354to Mozaffar describe various adsorbent resins and their use forunrelated products. These patents are only illustrative of the generalstate of the art in the use of adsorbent resins.

U.S. Pat. No. 5,503,867 to Pleva describes the use of whole groundcherries and oat bran in ground meat. The amount of cherries used was 10to 15% by weight and the oat bran is believed to be added to compensatefor the juice in the cherries. In any event, the cherries definitelycontribute a flavor to the meat and the palatability of the product isnot universally accepted.

Recent studies on stabilization of low-fat ground beef with cherrytissue suggest that this plant source contains potent antioxidants whichnot only suppress lipid peroxidation, but also inhibit formation ofheterocyclic aromatic amines and cholesterol oxidation products duringfrying (Gomaa et al., IFT Abstracts No. 68E-7 (1996). The hypothesisused to explain these observations was that polyphenols, such asflavonoids, anthocyanins and anthocyanidins, frequently found in thevacuoles of higher plants such as the cherries were responsible for thisantioxidant effect.

There is a need for natural edible berry derived compositions for use,particularly as dietary supplements/nutraceutical or food additives.

SUMMARY OF THE INVENTION

The present invention relates to a method for producing a mixturecomprising anthocyanins, bioflavonoids and phenolics from an edibleberry as a composition which comprises:

(a) providing an aqueous solution containing the anthocyanins,bioflavonoids and phenolics from the berry;

(b) removing the anthocyanins, bioflavonoids and phenolics onto a resinsurface from the aqueous solution;

(c) eluting the resin surface with a eluant to remove the anthocyanins,bioflavonoids and phenolics from the resin surface; and

(d) separating the eluant from the anthocyanins, bioflavonoids andphenolics.

Further, the present invention relates to a method for producinganthocyanins, bioflavonoids and phenolics from an edible berry as acomposition which comprises:

(a) providing a first batch of berry, wherein the cherries are fresh orquick frozen and thawed;

(b) disrupting the berry and separating pulp from the juice;

(c) extracting the anthocyanins, bioflavonoids and phenolics from thepulp into an aqueous solution;

(d) removing the anthocyanins, bioflavonoids and phenolics ontoadsorbent resin particles from the aqueous solution containing theanthocyanins, bioflavonoids and phenolics separated from the pulp;

(e) washing the resin particles with a lower alkanol to remove theanthocyanins, bioflavonoids and phenolics from the resin particles;

(f) separating the alkanol from the anthocyanins, the bioflavonoids andphenolics; and

(g) repeating steps (a) to (e) with the separated alkanol and the resinparticles from which the anthocyanins, bioflavonoids and phenolics havebeen removed with a second batch of the berry.

Further, the present invention relates to a consumable composition whichcomprises in admixture:

(a) dried mixture of isolated anthocyanins, bioflavonoids and phenolicsfrom an edible berry; and

(b) a food grade carrier, wherein the weight ratio of (a) to (b) isbetween about 0.1 to 100 and 100 to 0.1.

Finally, the present invention relates to a method for feeding a mammalwhich comprises:

feeding the mammal a consumable composition which comprises inadmixture:

(a) dried mixture of isolated anthocyanins, bioflavonoids and phenolicsremoved from an edible berry; and

(b) a food grade carrier wherein the weight ratio of (a) to (b) isbetween about 0.1 to 100 and 100 to 0.1. It is preferred that thecomposition contain at least in part dried pulp of the berry.

The term “anthocyanins” means the compounds that impart color incherries.

The term “bioflavonoids” means the isoflavonoids and flavonoid compoundscontained in cherries.

The term “phenolics” refers to compounds with a phenyl group and havingone or more hydroxyl groups from cherries.

Some of the edible berries are cranberry, raspberry, strawberry,blueberry, blackberry, elderberry, red grapes, gooseberry, Barbadoscherry (acerola cherry) and choke cherry.

OBJECTS

It is therefore an object of the present invention to provide a naturalsource edible berry composition which can be used in foods or as dietarysupplements or nutraceuticals. Further, it is an object of the presentinvention to provide a method for isolating the composition on acommercial scale. Finally, it is an object of the present invention toprovide a natural source composition which is economical to prepare andeasy to use. These and other objects will become increasingly apparentby reference to the following description and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the isolated anthocyanins (colorants) fromBALATON and MONTMORENCY cherries. The aglycone cyanidin has a hydroxylgroup at position 3.

FIGS. 2 and 3 are drawings showing the major bioflavonoids isolated fromthe cherries, as described in provisional application Serial No.60/111,945, filed Dec. 11, 1998.

FIG. 4 shows the phenolics isolated from tart cherries.

FIG. 5 shows the steps in the method of the present invention asdescribed in Examples 1 and 2.

FIG. 6 is a schematic drawing showing the use of an open vessel 10 forholding resin beads, which remove anthocyanins and phenolics from thecherry juice.

DESCRIPTION OF PREFERRED EMBODIMENTS

The cherries used in the present invention can be sweet or sour,although the latter are preferred since they contain high levels ofmalic acid in addition to other organic acids which contributes to thesour taste of tart cherries. The method of the present inventionisolates malic acid and other organic acids containing sugars which canbe used in foods to provide tartness and flavor. Most preferred are theBALATON and MONTMORENCY cherries.

The isolated mixture of anthocyanins, bioflavonoids and phenolics can betableted and used as a natural nutraceutical/dietary supplement. Ingeneral, the tablets provide a daily dose of the anthocyanins andbioflavonoids of about 1 to 200 mg, preferably a daily dose of 60-100mg. One hundred (100) cherries provide 60 to 100 mg of anthocyanins. Thephenolics (FIG. 4) are provided in an amount of 0.1 to 50 mg as a dailydose. One hundred cherries provide 1-50 mg of phenolics. The amount ofthe anthocyanins, bioflavonoids and phenolics can be adjusted byisolating the individual compounds and blending them together. It ispreferred to use the natural mixture of the anthocyanins, bioflavonoidsand phenolics which is isolated by the resin.

The resin has a surface to which the anthocyanins, bioflavonoids and thephenolics are adsorbed. A preferred class of adsorptive resins arepolymeric crosslinked resins composed of styrene and divinylbenzene suchas, for example, the AMBERLITE series of resins, e.g., AMBERLITE XAD-4and AMBERLITE XAD-16, which are available commercially from Rohm & HaasCo., Philadelphia, Pa. Other polymeric crosslinked styrene anddivinylbenzene adsorptive resins suitable for use according to theinvention are XFS-4257, XFS-4022, XUS-40323 and XUS-40322 manufacturedby The Dow Chemical Company, Midland, Mich., and the like.

It is preferred to use commercially available, FDA-approved,styrene-divinyl-benzene (SDVB) cross-linked copolymer resin, (e.g.,AMBERLITE XAD-16). Thus, in the preferred embodiment, AMBERLITE XAD-16,commercially available from Rohm and Haas Company, and described in U.S.Pat. No. 4,297,220, herein incorporated by reference, is used as theresin. This resin is a non-ionic hydrophobic, cross-linked polystyrenedivinyl benzene adsorbent resin. AMBERLITE XAD-16 has a macroreticularstructure, with both a continuous polymer phase and a continuous porephase. In a particularly preferred embodiment, the resin used in thepresent invention has a particle size ranging from 100-200 microns.

It is contemplated that other adsorbents such as those in the AMBERLITEXAD adsorbent series which contain hydrophobic macroreticular resinbeads, with particle sizes in the range of 100-200 microns, will also beeffective in the methods of the present invention. Moreover, differentvariations of the AMBERLITES, such as the AMERCHROM CG series ofadsorbents, used with particle sizes in the range of 100-200 microns,may also be suitable for use in the present invention. The AMBERLITEXAD-16 is preferred since it can be re-used many times (over 100 times)However, it is contemplated that for food, the use ofgovernmentally-approved resins in the present invention will beconsidered important and/or desirable.

Any solvent can be used to remove the adsorbed anthocyanins,bioflavonoids and phenolics. Preferred are lower alkanols containing 1to 4 carbon atoms and most preferred is ethanol (ethyl alcohol) since itis approved for food use. Typically the ethanol is azeotroped withwater; however, absolute ethanol can be used. Water containing malicacid and sugars in the cherries pass through the column. These arecollected and can be used in foods as flavors.

The anthocyanins, bioflavonoids and phenolics are preferably isolatedfrom the BALATON and the MONTMORENCY cherries. The composition of thecherries is in part shown in part by U.S. application Ser. No.08/799,788 filed Feb. 12, 1997 and in part U.S. application Ser. No.60/111,945, filed Dec. 11, 1998 which are incorporated by referenceherein.

The term “carrier” or “bulking agent” is used to mean a compositionwhich is added to increase the volume of the composition of the purifiedcomposition from the cherry. Preferred is dried cherry pulp. Theseinclude any edible starch containing material, protein, such as non-fatdry milk. Within this group are flour, sugar, soybean meal, maltodextrinand various condiments, such as salt, pepper, spices and herbs, forinstance. The bulking agent is used in an amount between about 10⁻⁶ and10⁶ parts by weight of the mixture.

The composition is introduced into the food in an amount between about0.1 and 10 mg/gm of the active ingredients of the food. The amount ispreferably selected so as to not affect the taste of the food and toproduce the most beneficial result. The food can be high (wet) or lowmoisture (dry) as is well known to those skilled in the art. When usedas a dietary supplement the tablets contain between 0.1 to 1 gram ofactive ingredient.

Methods have been developed for extraction and isolation ofphytochemicals (Chandra, A., et al., J. Agric. Food Chem. 41:1062(1992); Wang, H., et al., J. Agric. Food Chem. 45:2556-2560 (1997)) andfor rapid screening of antioxidant activity (Arora, A. and G. M.Strasburg, J. Amer. Oil Chem. Soc. 74:1031-1040 (1997)). These methodsare being utilized to identify and characterize the antioxidantcompounds from BALATON and MONTMORENCY cherries. Juiced cherry tissuewas sequentially extracted with hexane, ethyl acetate and methanol. Bothmethanol and ethyl acetate fractions showed strong antioxidant activityin the screening assay. The ethyl acetate fraction was further purifiedby silica gel vacuum liquid chromatography to yield four subfractions;the subfraction which showed the strongest antioxidant activity wasfurther separated into seven fractions by preparative reverse phaseHPLC. FIGS. 2 and 3 show the bioflavonoids isolated from the BALATONcherries. There are thus numerous analogous or homologous compounds inthe tart cherries.

Two novel phenolic compounds were identified: I) 1-(3′-4′-dihydroxycinnamoyl)-2,3-dihydroxy cyclopentane, and II) 1-(3′-4′-dihydroxycinnamoyl)-2,5-dihydroxy cyclopentane. Other compounds isolated from theethyl acetate extract of cherry fruits and characterized by spectralmethods include: 1-(3′-methoxy, 4′-hydroxy cinnamoyl) quinic acid,2-hydroxy-3-(21-hydroxyphenyl) propanoic acid, methyl2-hydroxy-3-(2′-hydroxyphenyl) propanoate, D(+)-malic acid, β-sitosterolad β-sitosterol glucoside. FIG. 4 shows some of the phenolics which wereisolated. The anthocyanin components obtained from the juice fractionalso have been identified and fully characterized (Chandra, A., et al.,J. Agric. Food Chem. 41:1062 (1992); Wang, H., et al., J. Agric. FoodChem. 45:2556-2560 (1997)); the results indicate that these compoundscontain potent antioxidant activity.

EXAMPLES 1 and 2

As shown in FIG. 5, individual quick frozen (IQF) cherries (which hadbeen pitted) were defrosted and blended in an industrial WARING blender.The mixture was centrifuged at 10,000 rpm and the juice was decanted.The residue, pulp, was further pressed with cheese cloth to remove anyadditional juice.

The pulp was lyophilized at 15° C. The juice was processed on AMBERLITEXAD-16 HP resin to produce cherry sour, anthocyanins, bioflavonoids andphenolics. The XAD-16 resin, 1 kg, was washed with ethanol (1-2 L) andthen washed with water (6 L). The XAD-16 resin was allowed to stand inwater for 1 hour before loading into a glass column (10 ID×90 cm long)with a cotton plug. The packed column was washed with water (2 L) beforeloading the juice for separation. 800 mL juice was purified each time.The juice was added onto the surface of the column and allowed to settlewith no flow. It was then eluted with water and the first 1 L wasdiscarded. The next 2 L of washing was collected, since it contained thecherry juice which was sour since it contained malic acid and sugarsfrom the cherries. The column was then washed with an additional 4 L ofwater in the case of BALATON and 5 L for MONTMORENCY cherry juice. Oncethe cherry juice was collected, the remainder of the washing with waterwere discarded. The column was then eluted with ethanol (1.3-1.5 L) andcollected the red solution containing anthocyanins, bioflavonoids andphenolics (700-800 ml). The column was then run dry and washed with 10 Lof water before repeating the process many of times (over 100).

The red alcoholic solution was then evaporated under vacuum a (20millitorr) to remove ethanol and the aqueous solution, stabilized with50 ppm ascorbic acid, was lyophilized at 10° C. The red powder wascollected and stored.

Example 1 results: BALATON cherry Weight of IQF cherries 15.74 kg Weightof dried pulp 605 g Volume of juice 12.16 L Weight of anthocyanins,bioflavonoids 31.35 g and phenolics (red powder) Volume of sourbyproduct @ 35 L (malic acid and sugars) Example 2 results: MONTMORENCYcherry Weight of IQF cherries 30.45 kg Weight of dried pulp 895 g Volumeof juice 24.03 L Weight of anthocyanins, bioflavonoids and 47 gphenolics (red powder) Volume of cherry by-product @ 75 L (malic acidand sugars)

The red powders of Examples 1 and 2 were preferably mixed with driedpulp as a carrier and tableted into 1 to 1000 mg tablets including thecarrier (1 adult daily dose).

Various food grade acids can be added to the isolated anthocyanins,bioflavonoids and phenolics to prevent decomposition. Preferably they donot add flavor. Ascorbic acid (vitamin C) is preferred. The acid can beadded before or after the drying of the cherry compounds.

For small scale processing, lyophilization is used to remove water. Forlarger scale production, drying in an air circulating oven is preferred.

EXAMPLE 3

As shown in FIG. 6, an open vessel 10 is provided with an inlet line 11and an outlet line 12, with valves 13 and 14, respectively. The resinbeads 15 are provided in the open vessel 10. Water is introduced intothe vessel 10 and then removed through outlet line 12 and discarded. Thecherry juice (without the pulp or pits) as in Example 1 is introduced tothe vessel 10 and allowed to stand for 25 minutes. The temperature ofthe water and juice is between about 20 and 30° C. The cherry juiceresidue containing malic acid and sugars is then removed through theoutlet line 12 and retained as a food flavoring. The resin 15 in thevessel is then washed again with water from inlet line 11 and thenremoved and discarded through outlet line 12. The anthocyanins,bioflavonoids and phenolics on the resin particles are then extractedusing 95% ethanol introduced through inlet line 11. The ethanolcontaining the anthocyanins, bioflavonoids and phenolics is removed fromthe vessel 10. The ethanol is removed from the anthocyanins,bioflavonoids and phenolics and dried using flash drying under nitrogen.The resulting powder is preferably then mixed with dried cherry pulp orother carrier as in Example 1. The resin particles are washed with waterand then the resins and ethanol are recycled many times.

EXAMPLE 4

Crude ethyl acetate extracts from cherries (containing anthocyanins,bioflavonoids and phenolics) were tested in aqueous solution undervarious conditions using a fluorescent assay for antioxidant activity.The fluorescent assay is described first.

Fluorescence assay for antioxidant activity (general): The need toscreen large numbers of compounds or extracts for antioxidant activityrequires that a model system (or systems) be employed which reasonablywell represents the structural and functional characteristics of thecomposition alone or in the food product. The test must also besensitive, rapid, and inexpensive. A fluorescence-based assay forevaluating antioxidant efficacy was used (Arora, A., and G. M.Strasburg, J. Am. Chem. Soc. 1996)). Large unilamellar vesiclesconsisting of 1-stearoyl-2-linoleoly-sn-glycero-3-phosphocholine wereprepared, which closely resemble the properties of biological membranes,one of the primary sites of peroxidation. A fluorescent probe,1,6-diphenylhexatriene propionic acid, is incorporated into themembranes such that the polar head group anchors the probe near theaqueous interface, while the hydrophobic portion lies parallel to thefatty acid chains. This probe reacts with the free radicals generatedduring peroxidation, resulting in a decrease in fluorescence intensitywith time. A peroxidation initiator (such as ferrous metal ions or thefree radical generator AAPH (Azobis-[2-amidino propane hydrochloride])is used to start the reaction, and the kinetics of fluorescence decreaseare determined in the presence or absence of the antioxidant compositionto be tested. An assay for a compound at a given concentration presentlytakes only twenty-one minutes, consumes only a few micrograms of lipid,and can be readily conducted with a simple fluorometer.

Large unilamellar vesicles (LUVs) were prepared from1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine according to theprocedure outlined by MacDonald et al (MacDonald, R. C., et al.,Biochim. Biophys. Acta 1061:297-303 (1991)). Briefly, the lipid wasdissolved in chloroform, and was dried to a thin film using a rotaryevaporator. The dried film was resuspended in an aqueous buffer, and wasrepeatedly extruded through a polycarbonate filter of 100 nm pore sizeusing a Liposofast piposome extruder (Avestin, Inc., Ottawa, Canada).The homogeneity of size (80-100 nm) and the unilamellar nature of thevesicles were confirmed using freeze-fracture scanning electronmicroscopy. The fluorescent probe, diphenylhexatriene-propionic acid(DPH-PA), was incorporated into the vesicles during preparation at amole ratio of 1:350 (probe:lipid). For the fluorescence experiments,LUVs containing.DPH-PA is suspended at a final concentration of 100 μMin 100 mM NaCl, 50 mM tris-HEPES buffer at pH 7.0. The fluorescent probewas excited at 384 nm and emission was monitored at 423 nm. Lipidoxidation is inhibited in the LUVs by addition of ferrous ions or thefree radical generator AAPH; the progress was monitored by the decreaseof the fluorescence intensity of DPH-PA resulting from reaction withfree radicals generated over twenty-one minutes. A plot of the decreaseof fluorescence intensity as a function of time was used to determinethe kinetics of lipid oxidation. The results show that a mixture of thecrude anthocyanin extract with ethylacetate was effective in inhibitingoxidation.

Solvent extraction of the anthocyanins, bioflavonoids and phenolics canbe used; however this is not preferred where the product is to be usedas a food and for expense reasons. Where the preferred adsorbent resinsare used, this step is unnecessary. It is also possible to separate andrecombine the components using chromatography; however, for the purposeof the present invention, this is far too expensive since it involveshigh pressure liquid chromatography.

EXAMPLE 5

The compositions were tested for anti-inflammatory activity usingcyclooxygenase I and II (COX-I and COX-II) in an assay as described inWang et al., J. Nat. Products 62:294-296 (1999); Wang et al., J. of Ag.and Food Chemistry, 47: 840-844 (1999) and Wang et al., J. of Nat.Products, 62:86-88 (1999). The results were that the compositionsexhibited anti-inflammatory activities, specifically strong inhibitionof COX-I and COX-I.

EXAMPLES 6, 7 AND 8

Processing of Raspberries, Blueberries, and Blackberries:

Raspberries (339 g), Blueberries (350 g) and Blackberries (670 g) weredefrosted and blended separately with 500 mL of water in an industrialWaring blender. The mixtures were centrifuged at 10,000 rpm for 20minutes and juice was decanted. The residues (pulp) were further pressedwith cheesecloth to remove any additional juice. The juice aftercentrifugation was about 775 mL (Raspberries), 700 mL (Blueberries), and1100 mL (Blackberries).

The pulp was lyophilized at 15° C. The juices were processed on XAD-16resin to separate anthocyanins and phenolics from sugars and acids. TheXAD-16 resin (1 kg) was washed with ethanol (1-2 L) and then washed withwater (6 L). The resin was allowed to stand in water for 1 hour beforeloading into a glass column (10×90 cm) with a cotton plug. The packedcolumn was washed with water (2 L) before loading the juice forseparation. 800 mL of juice was purified each time. The juice was addedon to the surface of the column and allowed to settle with no flow. Itwas then eluted with water and the first 1 L was discarded. The next 2 Lof washing was collected, since it contains the sugars and acids. Thecolumn was then washed with an additional 3 L of water for all juices.After removing sugars and acids, the column was eluted each time withethanol (1.3 L, each for Raspberries and Blueberries and 1.0 L forBlackberries) and collected the red solution containing anthocyanins andphenolics (700 mL). The column was then run dry and washed with 10 L ofwater before repeating the process.

The red alcoholic solution was then evaporated under vacuum to removeethanol and the aqueous solution, stabilized with 50 ppm ascorbic acid,was lyophilized at 10° C. The red powders were collected and stored at−20° C. The results are shown in Table 1.

Water Volume of Volume added Volume of water needed of EtOH Weight ofWeight of to make juice after to remove used to elute residues afterAnthocyanins weight juice centrifugation acids and Anthocyaninslyophilization and phenolics Fruit (g) (mL) (mL) sugars (mL) andphenolics (mL) (g) (g) Raspberries 339.0 500 775 6000 1300 13.76 0.6752Blueberries 350.0 500 700 6000 1300 52.20 0.7110 Blackberries 670.0 5001100 6000 2000 51.65 1.4420

It is intended that the foregoing description be only illustrative ofthe present invention and that the present invention be limited only bythe hereinafter appended claims.

I claim:
 1. A consumable composition with antioxidant andanti-inflammatory activity which comprises in admixture: (a) a driedmixture of isolated anthocyanins, bioflavonoids and phenolics from anedible berry free of acids and sugars of the berry and berry solidswherein the mixture of anthocyanins, bioflavonoids and phenolics areprovided in an amount that is effective to provide antioxidant andanti-inflammatory activity and the mixture includes at least onephenolic selected from the group consisting of

where R is selected from the group consisting of H and CH₃; and

wherein R₁ is OH and R₂ is H or R₁ is H and R₂ is OH; and (b) a foodgrade carrier wherein the weight ratio of (a) to (b) is between about0.1 to 100 and 100 to 0.1.
 2. The composition of claim 1 wherein thecarrier is a dried pulp of a cherry.
 3. A method for providingantioxidant and anti-inflammatory activity to a mammal which comprises:feeding the mammal a consumable composition with the antioxidant andanti-inflammatory activity which comprises in admixture (a) a driedmixture of isolated anthocyanins, bioflavonoids and phenolics from anedible berry free of organic acids and sugars of the berry and berrysolids, wherein the mixture includes at least one phenolic selected fromthe group consisting of

where R is selected from the group consisting of H and CH₃; and

wherein R₁ is OH and R₂ is H or R₁ is H and R₂ is OH; and (b) a foodgrade carrier wherein the weight ratio of (a) to (b) is between about0.1 to 100 and 100 to 0.1.
 4. The method of claim 3 wherein the carrieris a dried pulp of a berry.
 5. The method of claim 3 wherein the mammalis human.
 6. The method of claim 3 wherein the dried mixture of isolatedanthocyanins, bioflavonoids and phenolics is prepared by (a) providingan aqueous solution containing juice from the berry; (b) removing theanthocyanins, bioflavonoids, and phenolics from the organic acids andsugars of the berry and the berry solids in the solution by adsorbingthe anthocyanins, bioflavonoids, and phenolics onto a resin which doesnot adsorb the organic acids and sugars of the berry and the berrysolids, wherein the resin is certified for use with food products; (c)eluting the anthocyanins, bioflavonoids, and phenolics from the resinwith an eluant to produce the mixture of the anthocyanins, bioflavonoidsand phenolics in the eluant; (d) separating the eluant from the mixture;and (e) drying the mixture.
 7. The method of claim 3 wherein the driedmixture of isolated anthocyanins, bioflavonoids and phenolics isprepared by (a) providing a batch of the edible berries, wherein theberries are fresh or quick frozen and thawed; (b) blending the berriesand separating pulp from juice; (c) extracting the anthocyanins,bioflavonoids and phenolics with the organic acids and sugars of theberry and the berry solids into an aqueous solution from the juice andpulp; (d) removing the anthocyanins, bioflavonoids, and phenolics fromthe organic acids and sugars of the berry and berry solids in thesolution by adsorbing the anthocyanins, bioflavonoids, and phenolicsonto adsorbent resin particles which do not adsorb the organic acids andsugars of the berry and the berry solids, wherein the resin particlesare certified for use with food products; (e) washing the resinparticles with a lower alkanol to remove the anthocyanins, bioflavonoidsand phenolics as a mixture from the resin particles; (f) separating thealkanol from the mixture; (g) repeating steps (a) to (e) with theseparated alkanol and the resin particles from which the mixture hasbeen removed with multiple batches of the berries; and (h) drying themixture.
 8. The method of claim 7 wherein the alkanol is ethanol.